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Response to Reviewers # # Reviewer #1
We thank Reviewer #1 for the positive assessment, in particular for recognizing that the sex-independent role of PAGE4 is clearly supported by multiple layers of protein evidence, that the proteomic, phosphoproteomic and interactomic techniques are conducted at an expert level, and that the reporting of results is appropriate. We have addressed the constructive points on outlier testing and methods completeness in full.
Reviewer comment: Figure 1B: the first PCA component is dominated by a single sample far to the right (PC1 33.9%). Was an outlier test (Mahalanobis distance, Z-score on PC1) performed? If so it should be reported. No PCA or outlier test is reported for the phosphoproteomics. The full-proteome and phosphoproteomics data need more analysis detail to ensure reproducibility.
__Response: __We agree, and we have addressed this in two ways. First, we have replaced the Figure 1B proteome PCA with multidimensional scaling (MDS) computed from the top 500 most variable proteins, which displays the paired tumor-myometrium structure directly and is not dominated by a single component; the inputs are stated in the Methods and the Figure 1B legend: MDS was computed from the top 500 most variable proteins using log2-transformed intensities, with no imputation of missing values. Second, to address the reviewer's underlying concern about undetected technical outliers, we inspected the per-sample distribution of log2 protein intensities for every sample in the DIA cohort. One HMGA2-subtype tumor measurement was clearly aberrant at the technical level: its distribution is bimodal and its median is collapsed (~2.9 versus a cohort median of 7.8), the signature of a sample-specific quantification failure rather than biological variation (rebuttal figure below, panel A), whereas the same sample is unremarkable in the phosphoproteome (panel B), showing that the defect is specific to that one proteome measurement and not a property of the tumor. That measurement is not part of the dataset reported in the manuscript: the DIA cohort as analyzed comprises 28 matched tumor–myometrium pairs (56 samples), which is what the Methods, the figure legends and Supplementary Dataset 2 describe throughout. Every remaining sample passes this inspection, so no result reported here rests on a sample with a failed proteome measurement. We provide the per-sample distributions here for the reviewer's inspection rather than in the manuscript, since they document the composition of the analyzed cohort rather than adding to its findings.
Rebuttal figure1 for reviewer inspection only. Per-sample intensity distributions across the DIA cohort. (A) Proteome; (B) phosphoproteome. Each box is one sample's distribution of log2 intensities; the blue dashed line marks the cohort median (7.8). The aberrant sample (red) is a clear technical outlier in the proteome but unremarkable in the phosphoproteome; it is not part of the 28-pair dataset reported in the manuscript.
Reviewer comment: Figure 3A: perform an outlier test for HMGA2 (proteome, left) and for the FH_UL and COL4A5-COL4A6_UL points (phosphosites) that are visually separated. As shown, the PCAs are hard to interpret because points get squeezed onto one axis. Outliers may affect statistics and the conclusions about within-group variation; this should be tested if discussed.
Response: We have addressed both points. The same per-sample inspection described above covers every sample contributing to the Figure 3A proteome, phosphoproteome and phosphosite analyses, and all 28 pairs shown there pass it. The COL4A5-COL4A6 UL and FH UL samples highlighted by the reviewer are technically sound on these metrics and are retained; the text now attributes their separation to inter-tumor heterogeneity in phosphorylation state on the basis of these metrics rather than by assertion, which resolves the biological-versus-technical ambiguity the reviewer identified. Regarding axis compression, we agree that the phosphosite panel is stretched by a small number of samples with genuinely divergent phosphorylation profiles. Because these samples are technically sound, we have retained them rather than rescaling or trimming the axes, which would misrepresent the spread of the data; the proportion of variance explained by each component is stated on the axes so that the scale of the separation can be judged directly. The conclusions drawn from Figure 3 concern the tumor-myometrium contrast within each subtype, which is unaffected by the position of these individual samples.
Reviewer comment: SNRNP70_S226 is described as one of the most significantly hyperphosphorylated sites, but Table S1 lists log2FC 5.92, p 0.02, classified non-significant; MARCKSL1 (log2FC 2.14, p 0.016) and NDRG2 are likewise non-significant. The volcano/caption indicate a 0.05 cutoff but Table S1 apparently uses 0.01. Please clarify.
Response: We thank the reviewer for catching this, and we apologize for the inconsistency. On re-checking the source data we confirmed that the significance-classification column in the earlier version of the supplementary table had been generated with a stricter cutoff than the one used for the volcano plots and stated in the captions. All of the sites raised by the reviewer meet the stated criteria (|log2 fold-change| > 2 and p ##
Reviewer comment: Add citations for Protein Atlas and ProteomicsDB.
__Response: __Added: Human Protein Atlas (Uhlén et al., Science 2015; proteinatlas.org) and ProteomicsDB (Schmidt et al., Nucleic Acids Res 2018).
__Reviewer comment: __PAGE4 expression is selectively enriched in MED12-mutant UL (page 14): "PAGE4 expression indeed was significantly elevated in MED12-mutant ULs ... ". The figure caption, methods and text do not provide any information of the statistical test used regarding the statement of significant upregulation. Please provide the data or change wording as a statistical significance is implied.
Response: The panel reproduces published RNA-seq data (Berta et al., 2021) descriptively, and the source data do not provide a formal cross-subtype test that we can report. We have therefore softened the wording from “significantly elevated” to “consistently higher” in the Results, and the caption now states that the panel is descriptive and that no statistical test was applied.
Reviewer comment: Method section "Liquid chromatography-mass spectrometry (LC-MS)" report the UniProtKB database download date to ensure reproducibility. Does the database contain isoforms and TrEMBL sequences or uses a filtered version (e.g.: status reviewed, canonical?) (report it like on p.36 for the AP-MS/BioID samples). Further add a statement if missing values were imputed on which level (peptide, protein), which normalization strategy was employed, and was there filtering of incomplete values performed. All these analysis steps are common practice for DDA measurements of clinical samples and may affect subsequent statistical finding and thus should be reported in the method section if they were or were not performed (for the PPI data this was reported on p.37, but for the full proteome and phosphoproteome the information provided is not sufficient to judge the validity of data processing).
Method section "GO enrichment and statistical analysis" (p.37): Please add citations for the used tools SAINT, CRAPome and Enrichr.
__Response: __We have expanded the discovery DDA proteome and phosphoproteome Methods to the same level of detail as the AP-MS/BioID section: the UniProtKB human database version, download date and entry count; whether isoforms/TrEMBL were included or a reviewed-canonical subset was used; the valid-value filtering rule per group; the normalization strategy; and the imputation method and level. The corresponding DIA workflow is now also described (see Reviewer 2-J). Also added references for SAINTexpress (Teo et al., J Proteomics 2014), CRAPome (Mellacheruvu et al., Nat Methods 2013) and Enrichr (Kuleshov et al., Nucleic Acids Res 2016).
Reviewer comment: The Figure 1c: The scale of the (Gene) count does not correspond with the circle sizes of the plot. It is thus not possible to judge the number of genes per category. This should be sized equally as else it is not possible for a reader to judge the number of genes per enriched terms.
__Response: __We have corrected the GO enrichment panel (Figure 1D) so that dot area is drawn to a single consistent scale with an accurate, labeled size legend, allowing the gene count per term to be read directly.
Reviewer comment: Figure 1G: Expression levels for ProteomicsDB. As the log2 of the expression level is used, it is not clear if the white squares correspond to missing values in the ProteomicsDB, or if the value was very low. Maybe indicating absent values in a grey color scheme makes this more explicit.
__Response: __We have clarified this in the caption rather than by recolouring, because in this figure white already carries a defined meaning in both panels: in the Human Protein Atlas panel it is the explicit “not detected” category of the key, and in the ProteomicsDB panel the log2 intensity scale begins at zero, so a white cell corresponds to a value of zero, that is, no detected expression, rather than to an absent measurement. The caption now states this for both panels, so a white square can no longer be confused with a very low value.
Reviewer comment: Figure 1C: the caption indicates a "gray box" but it seems that PAGE4 was highlighted as a red dot, and a white box was added for indicating the gene name.
__Response: __The caption has been corrected to match the figure (PAGE4 shown as a highlighted point with a labeled callout), and color terminology has been harmonized across all captions.
Reviewer comment: Figure 3D is described as a volcano plot but is a horizontal scatter with a significance threshold rather than a classic volcano (which would have adjusted p-value on the axis).
Response: The reviewer is correct. These panels display log2 fold-change per subtype with significance indicated by color and do not plot a p-value axis, so “volcano plot” was the wrong term. We have relabeled them as grouped dot plots in the figure legend and at the two places they are referred to in the Results. Figure 2C and Figure 5A, which are conventional volcano plots with a -log10 p-value axis, retain that description.
Reviewer comment: The PPI network has too many edges and nodes to read. It could help to collapse categories/proteins or provide a subnetwork, with the full network in the supplement.
__Response: __We have moved the dense, full node-level PAGE4-centered network out of the main figure and into the supplement, where it now appears as Figure S3A. The main figure retains a complex-level view of these interactions as a focused interaction matrix (revised Figure 4E), in which preys are grouped by annotated functional complex with confidence-weighted encodings. This is the same change requested by Reviewer 3 (point G).
Reviewer #2
We thank Reviewer #2 for the positive overall assessment, that we convincingly identified a novel role for PAGE4 in uterine leiomyoma and Mediator transcriptional processes, that this represents an important contribution to understanding UL pathobiology, and that the dataset is very comprehensive. We are grateful for the detailed, panel-by-panel reading and the constructive suggestions on figure design and methods organization, all of which we have adopted.
Reviewer comment: The validation cohort is not technically a validation cohort, it uses the same 9 MED12-mutant UL samples and so cannot validate PAGE4 upregulation/phosphorylation in an independent cohort. A second MED12-mutant UL cohort should be used.
__Response: __We thank the reviewer for raising this, and we apologize that our original Methods wording was ambiguous. The discovery DDA, and validation DIA analyses were in fact performed on independent sets of MED12-mutant UL/myometrium samples (n = 9 each), with no patient shared between the sets; we originally sized (MED12-mutant) the DDA/IHC and DIA sets to match the nine discovery pairs, and then also had the opportunity to analyse also the other mutations causing UL. The DIA experiment therefore does provide independent validation of PAGE4 upregulation and Thr51/Thr85 phosphorylation in a separate cohort of MED12-mutant tumors, while additionally establishing subtype specificity against the HMGA2, FH and COL4A5-COL4A6 subclasses. We have revised the Methods to state the cohort composition explicitly so that the independence of the discovery (DDA) and validation (DIA) MED12 sample sets is unambiguous, and independent confirmation is further supported by the external RNA-seq dataset (Berta et al. 2021) and the IHC validation.
Reviewer comment: No meaningful change in peak width or height is readily observable. Statistical tests would be required to make the claims believable.
__Response: __We agree, and we have replaced visual assertions with quantification and statistics, reporting effect sizes alongside p-values. We now distinguish signal amplitude (which changes) from peak shape (which does not).
Amplitude is reduced (supported). Across a common set of 19,942 GENCODE v38 protein-coding TSSs, median promoter-proximal RNAP II signal (TSS ±1 kb) decreased by 10.4% in MED12 G44D, 16.5% in PAGE4 S9D/T51E/T85E and 21.2% in PAGE4 S9A/T51E/T85A relative to the matched wild type; the area under the curve decreased by the same margins, and median peak height (TSS ±250 bp) decreased by 4.4%, 20.4% and 19.3% respectively. All reductions are statistically significant (two-sided Wilcoxon rank-sum test, Benjamini–Hochberg-adjusted p Shape is unchanged (we concede this). Median TSS-derived FWHM was 201 bp in all five conditions (Δ = 0); the very small FWHM p-values arise from the large number of regions tested (n ≈ 17,000) and do not indicate a biologically meaningful shape change. MACS2 peak-width medians shifted inconsistently (+23 bp for G44D, +1 bp for S9D, −49 bp for S9A), i.e. no consistent broadening or narrowing.
We removed the statements that all four metrics concord and that peaks are narrower/broader. The revised Results now state that promoter-proximal RNAP II signal amplitude is modestly reduced, whereas peak shape (FWHM and MACS2 peak width) shows no consistent change, and we report effect sizes (median % change) for every metric. The ChIP-seq analysis pipeline (alignment, MACS2 peak calling, normalization, quantification) has been added to Methods, which previously lacked it.
Reviewer comment: HIPK2 was proposed as the candidate kinase (Fig 3E) but there is no HIPK2 on the heatmap; instead CLK2 is in red with an increased score. HIPK2 and CLK2 are different, unrelated kinases. Please rectify.
__Response: __We apologize for the inconsistency, which our cross-audit confirmed. We have replaced the kinase-family enrichment heatmap (original Figure 3E) with a per-site kinase-prediction map for PAGE4 S9, T51 and T85, now shown as the lower panel of Figure 4A, and revised the text so that figure and text agree. Both sites are proline-directed (S/T-P) CMGC substrates; the CLK and HIPK families score highest, with CLK2 and HIPK1 the most probable kinases, as both have been experimentally validated as PAGE4 kinases at these sites (Kulkarni et al., 2017; HIPK1 also reported at T51). We no longer single out HIPK2: motif scoring cannot resolve closely related paralogs, so HIPK2/HIPK3 and other CLKs remain possible but are not named individually, while the prior experimental literature points to HIPK1. The prediction is now explicitly framed as in silico and requiring direct validation (in vitro kinase assays with recombinant CLK2/HIPK1, and inhibitor or genetic perturbation).
Reviewer comment: The text states Mut1 retained 73% of PAGE4-WT prey proteins, but Fig 4B shows Mut1 with 25 preys vs 149 for WT, 25/149 = 17% retained, an 83% decrease. Please rectify.
__Response: __The reviewer is correct, and the original 73% was an error. We recomputed AP-only interactor retention directly from the final filtered interactor table (Supplementary Dataset 3). Defining retention as the fraction of PAGE4-WT AP high-confidence interactors (gene-level, n = 149) that are also recovered for Mut1, only 20 are shared, a retention of 13% and a net loss of 87% of stable associations; Mut1 captured 25 AP high-confidence interactors in total (bar plot, revised Figure 4C). The text and the Figure 4C legend now report these values consistently, and the statement about which categories are preferentially lost versus retained is tied to Supplementary Dataset 3 rather than asserted.
Reviewer comment: The text states the MED12-G44D bait is strongly reduced vs MED12 WT (Fig 5A), but the Fig 5A right panel does not support this. Please rectify.
__Response: __We thank the reviewer, on checking the data, the original statement was in fact wrong in the opposite direction, and we have corrected the text. Quantifying MED12 bait recovery directly from Table S3, the MED12-G44D bait is recovered at levels equal to or higher than MED12-WT (average spectral counts: AP-MS 519 vs 339; BioID 481 vs 191), not reduced. Despite this comparable-to-higher bait abundance, MED12-G44D captures markedly fewer high-confidence interactors (AP-MS 38 vs 115; BioID 297 vs 362). The Results now state this explicitly: the G44D mutation reduces MED12's high-confidence interaction repertoire while the bait itself is well expressed, so the loss of interactors is a genuine effect rather than a bait-recovery artifact. Figure 5A is a differential-interactor volcano (mutant vs WT) in which the bait's own abundance is not directly legible, which is why it appeared not to support the now-removed reduction claim. Bait spectral counts for every bait and both acquisition modes are reported in Supplementary Dataset 3, so bait recovery can be checked directly for each construct. In the interest of full transparency we note that the same check gives a different answer for two of the PAGE4 constructs: Mut1 and Mut6 are recovered at roughly ten-fold lower spectral counts than PAGE4 WT in both modes. We have therefore added an explicit limitation to the Discussion stating that reduced construct abundance or stability may contribute to the loss of stable interactors and to the compartmental shift observed for these two variants, and we have tempered the corresponding claims.
Reviewer comment: The text states MUT1 has upregulated base-excision repair and SUMOylation, but the Fig S3 heatmap supports this for MUT6, not MUT1. Please rectify.
__Response: __Thank you. We re-examined the pathway enrichment heatmap, which is Figure S4 in the revised manuscript, and confirm the reviewer is correct: base-excision repair, translesion synthesis and SUMOylation of DNA damage response proteins are enriched for the S9A/T51E/T85A variant (Mut6), not for Mut1. The Results text has been corrected so that text and figure agree.
Reviewer comment: Figure 4E shows the full bait-prey graph and becomes a hairball; bait names are illegible, edge widths/colors and AP/PL/Both cannot be distinguished. Suggest aggregating preys by annotated complex with a single summarized weighted edge per bait-complex, and showing only differentially associated preys; provide the full network in the supplement.
__Response: __We agree. The dense, full node-level network that produced the hairball has been moved out of the main figure and is now provided as a supplementary figure (Figure S3A) with a clearly keyed AP/PL/Both encoding. In the main figure, the corresponding interactions are presented at the level of annotated complexes as a focused interaction matrix (revised Figure 4E), which avoids the unreadable node-level graph while preserving the key bait-complex relationships.
Reviewer comment: The figure presents PAGE4 phosphosites but is labeled “GAGE,” a different (unused) name; importantly the figure appears copied directly from PhosphoSitePlus without permission. Permission should be obtained.
__Response: __We thank the reviewer and have resolved both issues. First, we replaced the PhosphoSitePlus-derived schematic with an original figure generated by us from the underlying site annotations, so no third-party copyrighted image is reproduced, removing the permissions concern entirely. Second, the “GAGE” label is showing the GAGE domain (PAGE4 belongs to the GAGE/PAGE family); all panel labels now read PAGE4 consistently, with the alias noted once in the text.
Reviewer comment: Inferring phosphorylation involvement from phosphomimetic changes is not formally valid and should at least be stated as a hypothesis.
__Response: __We agree and have reframed accordingly. We now state explicitly that phosphomimetic (S/T→D/E) and phosphodead (S/T→A) substitutions approximate the charge state of (de)phosphorylated residues but do not reproduce native, dynamic phosphorylation; the phosphorylation-dependence conclusions are therefore presented as a hypothesis supported by, but not proven by, these surrogates.
Reviewer comment: The MS methods are insufficient to reproduce the experiments. DIA methods are missing entirely; with both DDA and DIA used across total, phospho, AP and PL experiments, the methods should be reorganized by experiment type, specifying what was done in each mode. State how total and phospho data were normalized and integrated; how PL data were analyzed and whether identically to AP; whether SAINT used spectral counts or intensities; and, since the baits are nuclear, whether NLS-containing controls were used.
__Response: __We have substantially expanded and reorganized the MS Methods by experiment type and acquisition mode. (1) DIA acquisition and analysis for the validation proteome and phosphoproteome are now fully described, instrument, gradient and window scheme, search/quantification software and settings, library strategy, FDR and normalization. (2) For each dataset we state the acquisition mode: discovery proteome and phosphoproteome = DDA (Q-Exactive); validation proteome and phosphoproteome = DIA-PASEF (timsTOF Pro); AP-MS and PL-MS = DDA-PASEF (timsTOF Pro/Pro 2); host-cell-line proteome = DIA on an Orbitrap Astral, for which the full acquisition and DIA-NN parameters, including database version, entry count and the absence of isoforms, TrEMBL entries and imputation, are now given in Methods. (3) Total- and phospho-proteome integration: phosphosite intensities were normalized to matched parent-protein abundance for the protein-adjusted, site-level testing, and we describe the normalization, batch handling and integration of the two layers. (4) AP vs PL: we state explicitly that AP and PL data were processed through the identical pipeline (QRILC imputation → median normalization → SAINTexpress → CRAPome filtering), noting any differences. (5) SAINT input: we clarify that SAINTexpress was run on spectral counts (and CRAPome filtering used average spectral-count fold-change ≥ 3); the QRILC/normalization step applies to the quantitative interactor comparisons, and the previous ambiguity between counts and intensities is removed. (6) Nuclear-bait controls: GFP-MAC3 served as the negative control, and compartment-matched contaminants (nucleolar/chromatin) are additionally controlled by CRAPome frequency filtering; we discuss this explicitly and note its limitation for nuclear-bait specificity.
__Reviewer comment: __Overall the manuscript has many issues with data presentation. To illustrate just a few:
- Fig. 3B and 3D are described as volcano plots. They are not volcano plots.
- Fig. 3B legend mentions a lower panel. There is no lower panel for Fig. 3B.
- Fig. 3A, please use different shape markers for the UL subtypes and suggest connecting with edges to thus support their text conclusions of separate groupings.
- For Fig. 3E the colors for the families and groups are not distinguishable (and the font is too small). Please rectify.
- Please label the units and scale for Fig. 6A.
- Please mention the MAC3 tag uses Ultra-ID to thus justify the 10 min labeling time.
- In the relevant protein and phosphoproteomic datasets and figures please mention the number of proteins and phosphosites obtained to thus allow an interpretation of the data quality.
-Table 4 is in a format that is uninterpretable.
- The text and figures interchangeably use the mutant number (e.g. MUT1) or mutant composition (e.g. S9D_T51E_T85E - for MUT1), this makes reading and interpreting the manuscript very challenging. Suggest using one format.
__Response: __We have addressed each item:
- Fig 3B and 3D “volcano” plots: corrected. Both panels are now labeled grouped dot plots in the figure legend and in the Results, matching our response to Reviewer 1; Figure 2C and Figure 5A, which do plot a -log10 p-value axis, retain the term volcano plot.
- Fig 3B legend “lower panel” with no lower panel: the legend conflated the protein panel (3B) with the phosphosite panel (3D); the caption now matches the actual panels.
- Fig 3A subtype markers: we have retained the colour-coded points. Each of the eight groups already has its own colour and the legend states the group and n for each, and the eight-level palette was chosen so that each subtype's tumour and myometrium share a colour family, which is the comparison the panel is meant to support. We considered adding shape markers and 95% confidence ellipses, but at these group sizes (n = 5 for two subtypes) an ellipse conveys more confidence in the grouping than the data warrant, and connecting edges between unpaired samples would imply a trajectory that does not exist. The panel is therefore presented as a descriptive overview of sample structure; the subtype and tumour-versus-myometrium differences that the manuscript actually concludes on are tested statistically and reported in Figure 3B and Supplementary Dataset 2, not inferred from visual separation in the PCA.
- Fig 3E colors/fonts: the original Figure 3E no longer exists; it has been replaced by the site-level kinase-prediction panel that is now the lower panel of Figure 4A, which uses a larger font and a distinguishable family/group palette.
- Fig 6A units/scale: the Figure 6A legend now specifies that the heatmap shows MACS2 fold-enrichment (FE) signal over local background (macs2 bdgcmp, FE mode), in 50-bp bins across ±5 kb from GENCODE v38 protein-coding TSSs, with the display color scale capped at 20 FE units (values above 20 capped only for visualization; quantitative analyses used uncapped values).
- MAC3 labeling time: we now state that the MAC3 tag incorporates the UltraID biotin ligase, justifying the 10-minute labeling window.
- Dataset sizes: the number of proteins and phosphosites identified in the discovery datasets is stated in the Results, and the complete per-feature quantifications for both the discovery and validation datasets, from which the totals can be read directly, are provided in Supplementary Datasets 1 and 2.
- Table S4 formatting: reformatted into an interpretable layout.
- Mutant nomenclature: standardized to “Mut1 (S9D/T51E/T85E)” on first mention and “Mut1” thereafter. The full Mut1-Mut8 mapping is now stated explicitly in the Results at the point the panel is introduced, and the same convention is applied throughout.
Reviewer #3
We thank Reviewer #3 for the positive and thorough assessment, in particular for recognizing that we convincingly demonstrated the specific elevation of PAGE4 mRNA, protein and T51/T85 phosphorylation in MED12-mutant UL, that this points to a broader, sex-independent function for a traditionally male-specific marker, and that the work will be of significant interest to the UL research community. We address the major and minor points in turn.
Reviewer comment: The interactome, reporter and ChIP-seq studies relied exclusively on tagged, exogenously expressed proteins. Endogenous PAGE4/MED12 could substantially affect the observed networks. Measure endogenous expression in the Flp-In 293 T-Rex cells and discuss its influence. Did phosphovariants affect PAGE4 nuclear localization? Comparing T51/T85 phosphorylation between WT and G44D MED12 cells would be informative.
__Response: __We have characterized endogenous PAGE4 and MED12 in the parental Flp-In T-REx 293 line. By single-shot DIA proteomics of the parental cells on an Orbitrap Astral, PAGE4 was below detection, indicating that the tagged PAGE4 constructs are expressed against an effectively null endogenous background, consistent with PAGE4's restricted cancer-testis expression; the reported PAGE4 interactions, localization and reporter effects are therefore not confounded by an endogenous PAGE4 pool. MED12, an essential Mediator subunit, is endogenously expressed, so the tagged MED12-WT and MED12-G44D constructs are present alongside the endogenous protein, as we now note in the Discussion. We have also assessed whether PAGE4 phosphovariants alter nuclear/cytoplasmic distribution using our developed MS microscopy analysis (Liu et al, 2018, Nature Communications), We also agree that comparing PAGE4 T51/T85 phosphorylation between WT and G44D MED12 backgrounds would be informative; this requires phosphosite-specific reagents that we do not currently have, and we have flagged it explicitly in the Discussion as a priority for follow-up rather than claiming it here. The revised Discussion frames the heterologous overexpression system and its bearing on interpretation accordingly.
Reviewer comment: Turunen et al. (2014, PMID 24746821) reported mutant MED12 interactomes using a similar strategy. Comparing with this dataset would assess reproducibility and robustness and add confidence.
__Response: __We thank the reviewer for this excellent suggestion, and we note that a co-author of the present study also co-authored Turunen et al. (2014). We have compared our MED12 WT and G44D affinity-purification (AP-MS) interactors with the normalized spectral-count data of Turunen et al. (2014, Table S1), normalizing both datasets to the MED12 bait (see the rebuttal figure below). The two studies are concordant on the central finding: the G44D mutation specifically reduces association of the Mediator kinase (CDK) module while core Mediator is retained. In our data the bait-normalized G44D/WT ratio for CDK8 is 0.45 and for Cyclin C (CCNC) is 0.56 (Turunen et al.: 0.33 and 0.36, respectively), and CDK19 — the module member lost most completely in Turunen et al. (ratio 0.00) — was not detected as a high-confidence interactor of G44D in our AP-MS data. By contrast, core Mediator subunits are retained in both studies (median G44D/WT ratio 0.87 across 23 shared subunits in our data, versus 0.92 in Turunen et al.). Thus our MED12 G44D interactome independently reproduces the selective CDK8/Cyclin C uncoupling reported by Turunen et al. (2014), while our combined AP-MS/PL approach extends this to the PAGE4 variants and to transient/proximal associations. We have added a sentence to the Discussion noting this concordance; the comparison figure is provided here for the reviewer's inspection.
Rebuttal figure 2. Concordance of the MED12 G44D interactome with Turunen et al. (2014). Both datasets are normalized to the MED12 bait and expressed as the G44D/WT ratio. (A) Mediator kinase (CDK) module: CDK8 and Cyclin C (CCNC) are reduced in both studies; CDK19, the module member lost most completely in Turunen et al. (ratio 0.00), was not detected (n.d.) as a high-confidence G44D interactor in our AP-MS data. (B) Core Mediator subunits are retained in both studies (median G44D/WT 0.92 in Turunen et al., 0.87 in the present study). The selective reduction of the CDK module with retention of core Mediator reproduces the central finding of Turunen et al. (2014).
Reviewer comment: It is unclear whether the system is appropriate for ER-dependent transcription. Endogenous ERα/ERβ levels are not given, and estrogen signaling is ligand-dependent, yet no estrogen treatment appears to have been included. Without receptor expression and ligand stimulation, the estrogen-reporter results are hard to interpret.
__Response: __This is a valid concern. The estrogen reporter is a defined dual-luciferase construct in which tandem estrogen response elements (ERE) drive firefly luciferase, and it does not include a co-expressed estrogen receptor. Because HEK293 cells express very low to negligible endogenous ERα and the assays were performed without ER co-expression or 17β-estradiol stimulation, the readout reflects ERE-responsive promoter activity in a near-ER-null context by design, rather than bona fide ligand-dependent ER signaling. We now (i) report the endogenous receptor status directly: neither ESR1 nor ESR2 is detected among the 8,708 protein groups quantified in the parental Flp-In T-REx 293 line by Orbitrap Astral DIA (Supplementary Data 5), confirming an effectively ER-null background; and (ii) explicitly qualify the estrogen-reporter interpretation in both the Results and the Discussion. Repeating the assay with ERα co-expression and 17β-estradiol stimulation would be required for a ligand-dependent readout, and we agree this is the appropriate next experiment, but it falls outside the scope of the present revision and we have therefore limited our claims accordingly. The phosphorylation-dependent PELP1 association (PL-specific to Mut1) is presented as motivating, not demonstrating, an ER-signaling link.
Reviewer comment: Page 15, the authors described "Among individual kinases, HIPK2 was the top candidate for PAGE4-T51/T85, both sites fitting their known serine/threonine consensus motifs.". HIPK2 did not appear in the list of kinases in Figure 3E. Please clarify the data source and the criteria for the ranking.
__Response: __Reconciled as described for Reviewer 2-C: the kinase-family enrichment heatmap has been replaced by a site-level kinase-prediction panel for S9, T51 and T85 (lower panel of Figure 4A), figure and text name the same kinases, and the scoring method, ranking criterion and data source are stated in the caption and Methods.
Reviewer comment: Supplementary Figure 4, significance marks were missing.
__Response: __Significance annotations have been added to all panels of the luciferase figure, which is Figure S5 in the revised manuscript (Figure S4 is the pathway-enrichment heatmap). Each panel now shows the individual replicate values with the mean ± SD, and the legend states the test, the number of replicates, the comparator (all comparisons against PAGE4 WT) and the meaning of each asterisk level.
Reviewer comment: The biological significance of the enriched motifs is somewhat overinterpreted, since motif enrichment alone does not establish functional involvement. The presentation could be improved with a figure of top motifs, enrichment statistics and/or logos; e.g. “PAGE4 WT maintained AP-1-driven transcription, whereas phosphorylation induced an expanded motif repertoire” is hard to follow.
__Response: __We agree, and we have reworked the motif analysis to lead with effect size rather than statistical significance, correcting several over-statements in the original text.
__The p-values overstate the enrichment. __Across the five conditions, 129–172 of 440 known motifs pass q ≤ 0.05, but ~80% of these have fold-enrichment __The individually named transcription factors were over-called. __Most factors named in the original text reach statistical significance yet have fold-enrichment close to 1.0 (e.g. AP-1 1.03–1.11, E2F3 1.03, ETS1 1.07, FOXA1 1.03–1.09), i.e. no meaningful enrichment. We have removed these factor-specific claims and the “AP-1-driven transcription” / “expanded motif repertoire” framing.
CTCF is the one robust result. CTCF is the only motif with fold-enrichment ≥ 1.3 in all five conditions (range 1.38–2.29, peaking at 2.29 in PAGE4 S9D); its paralog BORIS is second-strongest (1.18–1.58). No other known motif exceeds 1.5-fold enrichment in more than one condition. We therefore name CTCF as the principal candidate regulatory motif, reported with effect sizes, and provide a summary figure of the top enriched motifs by fold-enrichment (Rebuttal Figure 3, below).
Rebuttal Figure 3. Top enriched known motifs (HOMER) by fold-enrichment across the five conditions. Colour intensity = fold-enrichment (% target / % background); grey dash = motif not detected in that condition; n.s. = not significant (HOMER Benjamini q > 0.05). CTCF is the only motif with fold-enrichment >= 1.3 in all five conditions.
Venn filter and counts (corrected). Consistent with the effect-size-first approach described above, the Venn diagrams in Figure 6E now show robustly enriched known motifs (HOMER Benjamini q ≤ 0.05 AND fold-enrichment ≥ 1.2); we recomputed the overlaps directly from the five HOMER outputs using this filter (union denominator). Corrected values — MED12: WT = 10, G44D = 18, union = 21, G44D-unique = 11 (52.4%), shared = 7 (33.3%); PAGE4: WT = 19, S9D = 12, S9A = 11, union = 25, shared by all three = 3 (12.0%), S9D-unique = 4 (16.0%), S9A-unique = 1 (4.0%). This supersedes our earlier q ≤ 0.05-only statement, which counted many weakly enriched motifs (fold-enrichment Reviewer comment: The Figure 6D data do not sufficiently support the statement that MED12 WT enriched for chromatin remodeling/transcriptional regulation, G44D for stress-associated functions, and phospho-PAGE4 for nucleosomal DNA binding and ribosomal interaction terms. Please clarify.
__Response: __We agree — the original panel over-interpreted small per-construct differences by assigning a distinct function to each construct. We have replaced the panel (now Figure 6G) with a corrected analysis that no longer makes per-construct functional assignments.
The new Figure 6G is a term-by-condition dot plot of Gene Ontology Molecular Function enrichment of the peak-associated genes (clusterProfiler, redundant terms reduced by rrvgo at similarity 0.9, Benjamini–Hochberg-adjusted p-values). It shows that all five constructs enrich for the same core terms: the most strongly enriched term in every condition is structural constituent of ribosome, and the other enriched terms (cadherin binding, rRNA binding, ribonucleoprotein-complex binding, ubiquitin-ligase-related terms) are likewise shared across conditions rather than unique to any one. No construct-specific functional classes were detected, consistent with RNAP II promoter occupancy at highly transcribed genes. The Results text and Figure 6G legend have been rewritten to describe this shared-core finding, and the earlier “differentially enriched across constructs” wording and the stale “Figure 6D” reference have been removed.
Reviewer comment: The legend says AP (light) vs PL (dark), but the figure shows AP green and PL yellow. And on the 73% statement, if the labels are correct, AP HCIs are 25 for Mut1 and 149 for WT, i.e. only 17% retained.
__Response: __The legend has been corrected to the actual colors used in the figure, and the retention value has been recomputed and corrected as described for Reviewer 2-D.
Reviewer comment: The data supporting “interactions with basal transcription factors and cell-cycle regulators were preferentially lost, while Mediator subunits and RNA-processing factors were selectively retained” are not readily apparent. Indicate the relevant figure/table.
__Response: __We now cite the specific evidence, the focused interaction matrix (revised Figure 4E) and Supplementary Dataset 3, and have added a categorized lost-versus-retained summary so the statement is directly traceable to the data.
Reviewer comment: Page 19, it is unclear how the statement that "In the MED12 p.G44D dataset, the bait (MED12-G44D) itself is strongly reduced compared with MED12 WT, consistent with decreased recovery of the mutant complex." is supported by the data presented in Figure 5A. Please clarify.
__Response: __Addressed as for Reviewer 2-E: the data show the MED12-G44D bait is recovered at levels equal to or higher than WT, so the original “bait reduced” statement was corrected; the reduced number of G44D interactors is retained as a genuine, bait-independent effect.
Reviewer comment: Page 19, reference is needed for "PELP1 is a well-established scaffolding protein that couples estrogen receptor signaling to chromatin remodeling and has been implicated in hormone-dependent tumor progression in breast and ovarian cancers."
__Response: __A reference for PELP1 as an estrogen-receptor coactivator/scaffold implicated in hormone-dependent tumors has been added.
Reviewer comment: For luciferase reporter assay: In the materials and methods section, the authors wrote "Data are presented as mean {plus minus} standard deviation (SD)", but in the figure legend, the authors wrote "Data represent mean {plus minus} SEM". Please clarify this discrepancy. In addition, please provide information regarding the promoter used in the luciferase assay plasmids, and replace "Firefly luciferase reporter plasmid containing the promoter of interest" with the specific promoter name.
__Response: __We have harmonized the error-bar reporting: the Methods and the figure legend now both state mean ± SD, with the same number of replicates given in each. We have also replaced “promoter of interest” with the specific response element for each pathway (cell cycle/pRb-E2F → E2F; p53/DNA-damage → p53; MAPK/ERK → serum response element/SRE; MAPK/JNK → AP-1; Wnt → TCF/LEF; estrogen → estrogen response element/ERE).
Reviewer comment: “Buyukcelebi K et al, 2023” is cited as both 2023a and 2023b; the same issue affects “Lin et al, 2018” and “Turunen et al, 2014.”
__Response: __We have consolidated the duplicated entries and made the year-suffix usage consistent for these and all other multiply-cited references. We also carried out a complete audit of the bibliography against the in-text citations: every citation now resolves to exactly one entry, no entry is duplicated, and no entry is left uncited. Several references that were missing from the list have been added, and two citations that could not be traced to a specific source were removed from a sentence that is adequately supported by the remaining reference. Prompted by this comment we also checked each citation against the statement it supports rather than only against the bibliography, and corrected several placements: two extracellular-matrix references have been moved onto the matrix sentence they actually support, a reference on the co-occurrence of MED12 and HMGA2 alterations has been moved to the sentence reporting concurrent MED12 mutations in the COL4A5-COL4A6 tumors, a PAGE4-specific reference has been added alongside the general androgen-receptor citation, and one claim in the Discussion has been narrowed to the extracellular-matrix and cytoskeletal remodeling that the cited work actually addresses.
Reviewer comment: “PAGE4 emerged … at protein, transcript, and IHC levels …”, IHC characterizes protein expression; did the authors mean phosphorylation levels?
__Response: __Corrected. IHC measured PAGE4 protein abundance and (cytoplasmic-predominant) localization; the sentence has been rewritten so the three independent layers are stated accurately (protein by MS, transcript by RNA-seq, and protein by IHC), without implying IHC measured phosphorylation.
Reviewer comment: “COL4A5-COL4A6 subclass showed upregulation of several extracellular matrix markers (WNT4, COL3A1, COL4A1, MMP2)”, it is unclear whether WNT4 should be an ECM marker.
__Response: __We agree and have reclassified WNT4 as a hormone/Wnt-signaling factor rather than an ECM component, and corrected the sentence so only bona fide ECM markers are grouped together.
